In recent years the use of stem cells in therapy has had widespread approval due to the successes obtained in the treatment of pathologies which until now were considered incurable.
However, the known methods for obtaining stem cells are long, laborious and expensive.
Pluripotent stem cells (PSC) are a source available not only for research but also for the creation of drugs and for transplants (Wagers A. J. et al., 2002; Griffith L. G. et al., 2002).
There are embryo and adult stem cells: the former derive from 8-day blastocysts, while the adult ones can be obtained mainly from bone marrow, adipose or muscular tissue, peripheral blood and the umbilical cord.
The definition of stem cell is constantly evolving and, at the moment, there is no general consensus or standard method to isolate them or identify them. For all these cells, embryonic (ES), and adult, both haemopoietic (HSC) and mesenchymal (MSC) (Kuwana M. et al., 2003), various genetic markers have been identified, of which some are common to many cell types (Condomines M. et al., 2006; Kang W. J. et al., 2006; Zhao Y. et al., 2003; Rabinovitch M. et al., 1976).
At the moment, research is more oriented toward the use of stem cells isolated from embryonic tissue, fetuses and the umbilical cord, but this is creating legal and ethical problems.
Above all, as of today the use of these cells has various counter-indications such as: risks of infection, risk of rejection if transplanted and, in some mammals such as horses, the development of teratomas.
To overcome these problems, in “in vivo” therapy it is known to use autologous stem cells, preferably isolated from bone marrow, adipose tissue or peripheral blood. Starting from adult stem cells there is a differentiation step “in vitro” (or “ex vivo”) of the stem cells in the cell line desired by means of specific factors of induction of the differentiation, and a subsequent transplant step “in vivo” of the differentiated cell line obtained. In these methods there is the disadvantage of rejection phenomena since the differentiated cells, reintroduced into the organism concerned, are not recognized as self-cells, inasmuch as they lose the self-recognition factors during the step of induced differentiation in vitro.
In man, taking the stem cells from peripheral blood entails their purification through a process called aphaeresis or leucoaphaeresis. The cells are extracted from the blood, collected and then inoculated into patients immediately after chemotherapy or radiotherapy.
In aphaeresis, which lasts from 6 to 8 hours, the blood is taken from the vein of an arm or a vein of the neck or chest, and made to pass through a machine that removes the stem cells. The blood, thus purified, returns to the patient, whereas the cells collected are preserved through refrigeration in liquid nitrogen (Condomines M. et al., 2006; Kang W. J. et al., 2006). This technique, apart from being painful, is also extremely stressful for the patient. Above all, the technique does not provide a real discrimination and/or purification of the stem cells circulating. The main known techniques for purification are:
use of growth factors or plate derivates (TGF-B, VEGF), but the economic costs of extracting these are prohibitive (Hou M. et al, 2006);
isolation of stem cells taken from the bone marrow, which allows to purify and hence use for therapy about 15% of cells contained in the extracted material;
isolation of stem cells from adipose tissue, which requires a prior surgical removal of considerable quantities of tissue from the donor and does not allow intravenous administration;
IGF-1 (insulin-like growth factor 1) known as Tendotrophin (Fiedler J. et al., 2006);
UBM (urinary bladder matrix): this is a derivate of the pig, containing cytokines (but not nucleate cells), which induce cicatrization of the wound but not the regeneration of the zone with the lesion (Zhang Y S et al., 2005).
Another known method is described by Zhao Y. et al., in the article “A human peripheral blood monocyte-derived subset acts as pluripotent stem cells” and in WO-A-2004/043990. This is a method to prepare stem cells deriving from monocytes, which comprises the steps of isolating monocytes from peripheral blood, putting them into contact with a mitogenic component and subsequently effecting the culture of the monocytes from peripheral blood in conditions suitable for the propagation of the cells.
This method, which initially requires a step of isolating the monocyte and then an expansion step in a culture mean, is very long, about 15-20 days, to obtain a significant number of stem cells, and does not allow to obtain pluripotent stem cells, that is, non specialized, suitable to be inoculated directly and after a short time into the patient.
Again in the framework of preparing stem cells from monocytes, the documents WO-A-2005/046570, WO-A-2007/131200 and WO-A-03/083092 are also known. However, since they have to carry out a preliminary purification of the blood in order to isolate only a cell fraction, that is, the monocytes, and a subsequent expansion in order to obtain the desired stem cells, the methods described in these documents take a very long time, again in the order of 15-40 days, in order to obtain an acceptable quantity of stem cells.
In the light of the above, there is an obvious need to perfect an expansion method, or division and purification of adult stem cells from easily accessible sources, particularly the blood, preferably peripheral blood, which also allows to obtain stem cells suitable for pharmacological treatment.
There is also an obvious need to start the production of stem cells from blood, preferably peripheral, in the shortest possible time, so as to be able to intervene promptly on the patient.
Purpose of the present invention is therefore to achieve a kit for the collection of blood, preferably peripheral blood, for the production of pluripotent stem cells, so as to allow a rapid start to the production of pluripotent stem cells.
The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.